Method and assay for detection of residues

ABSTRACT

Embodiments described herein include detecting an analyte in a low pH sample. Some embodiments include detection of multiple analytes in a sample utilizing a plurality of analyte binders and a control zone containing multiple control zone capture agents. In some embodiments, the multiple control zone capture agents capture a plurality of binders within one control zone.

This application is a Continuation of U.S. application Ser. No.15/460,594, filed Mar. 16, 2017, which claims the benefit of U.S.provisional application No. 62/309,572, filed Mar. 17, 2016, which isincorporated herein by reference in its entirety.

FIELD OF THE TECHNOLOGY

The present disclosure relates generally to binding assays and, moreparticularly, to systems, assemblies, and methods for detecting thepresence of one or more analytes in a sample having a low pH.

BACKGROUND

Antibiotic residues in foods are a major food safety concern. Healthissues include bacterial resistance to drugs and allergic reactions. Toavoid the impact of such health issues, food is tested worldwide forantibiotics and other contaminants. One type of test method uses what iscommonly known as a lateral flow test strip.

Lateral-flow test strips for detecting one or more analytes in a fluidsample may include a capture agent immobilized within a region of thetest sometimes referred to as a detection zone. Detection zones caninclude test zones and control zones. A typical capture agent hasbinding affinity for a substance that may be in the mobile phase of thetest strip. Lateral-flow tests in which the binding of a substance froma mobile phase to a capture agent generates a visible signal, that canbe interpreted visually or using a reader, such as a spectrophotomer,are well known in the art. Examples of such devices are described inU.S. Pat. No. 5,985,675, issued Nov. 16, 1999; and U.S. Pat. No.6,319,466, issued Nov. 20, 2001, and U.S. patent application Ser. No.10,289,089, filed Nov. 6, 2002 (based on U.S. Provisional Application60/332,877, filed Nov. 6, 2001) all of which are incorporated herein byreference.

Lateral-flow tests are widely used in the food products industry. Oneapplication is in testing dairy products. However, other fluids oftencontain characteristics that limit, or even prohibit, efficient use oftraditional lateral-flow tests. For example, citrus juices and oils maynot be compatible with conventional lateral-flow tests because of theirlow pH. Yet, these, and other low pH fluids, are subject to regulationby the United States Food and Drug Administration (FDA). For example,citrus juices are commonly treated with antibiotics to prevent citruscankers and other fungal infections. While the FDA has approved certainantibiotics, such as oxytetracycline and streptomycin, the FDA hasprohibited others, such as penicillin, and requires that citrus juicesand oils be tested for unsafe levels of certain beta-lactam antibiotics.It is desirable, therefore, to provide juice-testing personnel with auser-friendly test that can be analyzed with or without a reader and candetect multiple beta-lactams, such as penicillin, when present at orabove a threshold level.

Therefore, Applicants desire detection of residues in a low pH samplewithout the drawbacks presented by the traditional systems and methods.

SUMMARY

In accordance with the present disclosure, test strips and systems areprovided for the analysis in a sample. This disclosure provides improvedsystems, devices, assemblies, and methods that are convenient,efficient, and safe for the user, particularly when used to detect thepresence or absence of an analyte in a low pH sample.

In one embodiment, a method for detecting one or more analytes in asolution includes applying a binder to a test strip, the binderconfigured for generating a detectable signal and capable of combiningwith the analyte to form a binder-analyte complex; immobilizing, withinone or more test zones of the test strip, at least one test zone captureagents capable of capturing the binder; immobilizing, within a controlzone of the test strip, at least one control zone capture agent adaptedfor capturing the binder and the binder-analyte complex; providing asample having a pH less than 7; raising the pH of the sample by dilutingwith a buffer to form the solution; and adding the solution to the teststrip; and wherein one or more analytes in the solution, when present,are adapted to combine with one or more of the binders to form thebinder-analyte complex and wherein a comparison of the signal in thecontrol zone to the signal in the test zone provides a test result. Insome examples, the sample is a citrus juice. The pH of the sample may bebetween about 2 and about 5.

In another embodiment, an assembly for detecting an analyte in asolution, the assembly includes (a) a buffer comprising a mixture ofbovine serum albumin and sodium bicarbonate, said buffer added to acitrus juice sample and increasing a pH of the citrus juice sample,wherein the combination of the citrus juice sample and the buffer formsthe solution; (b) a binder configured for generating a detectablesignal, wherein the binder can combine with an analyte from the solutionto form a binder-analyte complex; and (c) a test strip configured toallow a test solution to flow, wherein the test solution contains eitheror both a binder and a binder-analyte complex, the test stripcomprising: (i) a test zone having immobilized thereon a test zonecapture agent that, when the binder has not formed a binder-analytecomplex, captures the binder, (ii) a control zone having at least onecontrol zone capture agent adapted to capture the binder, whether or notthe binder has formed a binder-analyte complex, and wherein capture ofthe binder at either the test zone or control zone results in adetectable signal and wherein a greater signal in the control zone ascompared to the test zone indicates a positive result.

Another embodiment of the disclosures is a lateral flow assay system fordetecting one or more analytes in a solution, the system comprising: (a)a buffer configured to increase the pH of a low pH sample to an optimalpH, wherein the combination of the low pH sample and the buffer formsthe solution; (b) a binder configured for generating a detectablesignal, wherein the binder being adapted to combine with an analyte fromthe solution to form a binder-analyte complex; and (c) a test stripconfigured to allow a test solution to flow, wherein the test solutioncontains either or both a binder and a binder-analyte complex, the teststrip comprising: (i) a test zone having immobilized thereon a test zonecapture agent that, when the binder has not formed a binder-analytecomplex, captures the binder, (ii) a control zone having at least onecontrol zone capture agent adapted to capture the binder, whether or notthe binder has formed a binder-analyte complex, and wherein capture ofthe binder at either the test zone or control zone results in adetectable signal and wherein a greater signal in the control zone ascompared to the test zone indicates a positive result.

In some examples, the low pH sample has a pH less than about 7. The lowpH sample may have a pH between about 2 and about 5, for instance thelow pH sample may have a pH of about 3.

In particular examples, the low pH sample is a citrus juice. Forinstance, the citrus may include lemon juice, lime juice, grapefruitjuice, orange juice, and the like. The buffer may comprise a mixture ofa carrier protein and a base. The carrier protein may comprise bovineserum albumin. The base may comprise sodium bicarbonate. The sodiumbicarbonate may comprise about 0.7M. The optimal pH may be about 7.

In some examples, the control zone capture agents have affinity to eachother. For instance, the control zone capture agent may comprise anantibody. The control zone capture agent may comprise an antibodybinding protein. The antibody binding protein may comprise protein A. Atleast one of the control zone capture agents may comprise an antibodyand another comprises protein A. The binder may comprise an antibody.The binder may comprise a multianalyte binder derived from a bacteria.The control zone capture agent may be immobilized on the control zonethrough attachment to a carrier protein. The carrier protein maycomprise bovine serum albumin. One of the control zone capture agentsmay comprise an antibody binding protein and another control zonecapture agent is an antibody to which the antibody binding protein hasaffinity, and wherein the antibody is applied to the test strip with thebinder, and flows, either with the binder or attached to the binder, tothe control zone. The binder may comprise a beta-lactam binder andwherein one of the control zone capture agents comprises an antibody tothe beta-lactam binder. The binder may comprise a beta-lactam binder andwherein one of the control zone capture agents comprises an antibody tothe beta-lactam binder. One of the plurality of binders may comprise amultianalyte binder with binding affinity for beta-lactam antibiotics.The multianalyte binder may comprise a beta-lactam binder fromGeobacillus stearothermophilus. One of the control zone capture agentsmay comprise an antibody to the multianalyte beta-lactam binder. One ofthe plurality of binders may comprise an antibody. The antibody maycomprise an antibody to penicillin. One or more unlabeled bindersunlabeled binders may reduce test sensitivity relative to the analyte towhich the unlabeled binder has affinity.

In particular examples, at least two of the control zone capture agentsmay be antibodies. At least two central control zone capture agents maybe antibodies and wherein each of the antibodies may have affinity to adifferent species of animal of binder. One of the control zone captureagents comprises a polyclonal antibody and the other may comprise amonoclonal antibody. Each of the binders may comprise an antibody from adifferent species of animal and at least one of the control zone captureagents comprises an antibody to one of those different species. At leastone of the control zone capture agents may comprise an antibody bindingprotein. The antibody binding protein may comprise protein A.

In some embodiments, detection of one or more contaminants in a low pHsample is accomplished by diluting with a buffer to optimize the pH ofthe sample, wherein the optimal pH for the sample may be about 7. Forexample, the buffer may comprise a mixture of a carrier protein and abase. The carrier protein may be bovine serum albumin in one example.The base may be a weak base, such as sodium bicarbonate. In one example,the concentration of sodium bicarbonate within the buffer is about 0.7M.Antigens, haptens and their antibodies, hormones, vitamins, drugs,metabolites and their receptors and binding materials, fungicides,herbicides, pesticides, plant, animal and microbial toxins, may bedeterminable using the present methods and apparatuses. Other analytesthat may be determinable by the disclosed methods and apparatusesinclude antibiotics, such as beta-lactams, cephalosporins, erythromycin,sulfonamides, tetracyclines, nitrofurans, quinolones, vancomycin,gentamicin, amikacin, chloramphenicol, streptomycin and tobramycin andtoxins, such as mycotoxins, such as aflatoxin and vomitoxin and drugs ofabuse, such as opioids and the like, as well as the metabolites thereof.

The test can include one or multiple binders each with binding affinityfor one or more analytes. The test can also include a test zone captureagent and one or more control zone capture agents with binding affinityfor the binders. In an example, two control zone capture agents are usedeach with affinity to a different binder. In another example, twocontrol zone capture agents are used and the two control zone captureagents also have binding affinity to each other.

An example described herein is a test for detection of beta-lactamantibiotics, including test strips sensitive to penicillin G,amoxicillin, ampicillin, ceftiofur, cephapirin and penicillin withsensitivity to each at or below safe level. Such a test can include abinder for beta-lactam antibiotics, for example a beta-lactam bindingprotein derived from Geobacillus stearothermophilus (sometimes referredto as Bacillus stearothermophilus) (“B.st.”) with affinity to multiplebeta-lactams including the target beta-lactams (“the BL binder”).Generally binders with affinity to multiple drugs are hereinafterreferred to as “multianalyte binders”.

The test can also include a binder with greater specificity for aparticular analyte as compared to a multianalyte binder, hereinafterreferred to as a “specific binder”, for example an antibody to anantibiotic such as penicillin (“penicillin binder”) to which themultianalyte binder, such as the BL binder, may not have adequatesensitivity. In such a test, a test zone can contain a capture agent forthe binder, for example representative antibiotics. When two binders areused, such as a multianalyte binder and a specific binder, a detectionzone can include capture agents for both, for example capture agentsimmobilized in separate test zones. Immobilization can be through use ofa carrier protein, such as BSA.

The test can also utilize two or more binders having sensitivity forunrelated analytes such as different families of antibiotics or toxins.

A control zone can be used for comparison to the one or more test zonesor as a signal that the test functioned properly and is complete. Acontrol zone can also include a capture agent. In an example, one of thecontrol zone capture agents includes an antibody binding protein such asprotein A, protein G or protein AG or recombinant forms of the same. Inanother example, one of the control zone capture agents includes anantibody, for example an antibody to a multianalyte binder immobilizedon the control zone prior to testing. It is also possible that anantibody to a multianalyte beta lactam binder is not immobilized in thecontrol zone and is instead combined with a beta lactam binder prior totesting and flows to the control zone for capture. In such an example,there is a single capture agent immobilized on the control zone prior totesting which can be an antibody binding protein.

When the control zone capture agents include an antibody bindingprotein, and an antibody, the capture agents may have affinity to eachother, and, therefore, if combined, may have binding to each other. Thecontrol zone capture agents need not, however, have affinity to eachother. For example, the control zone capture agents can include avariety of antibodies, receptors, binding proteins and the like, eachwith affinity to at least one of the analyte binders. Generally,however, when multiple test zones are employed, it is preferable to havea control zone be used for comparison to more than one test zone. Inthat way, one control zone can be used to compare to more than one testzone, thereby simplifying test result interpretation. For example, ifthere are two test zones one control zone can be used, or if there arefour test zones then two control zones can be used.

In some embodiments one test zone and one control zone are used tocapture one labeled binder, the control zone includes multiple captureagents, each with different affinity to a binder, such as each withaffinity to different parts of the binder.

The above summary was intended to summarize certain embodiments of thepresent disclosure. Embodiments will be set forth in more detail in thefigures and description of embodiments below. It will be apparent,however, that the description of embodiments is not intended to limitthe present inventions, the scope of which should be properly determinedby the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the disclosure will be better understood by a reading ofthe Description of Embodiments along with a review of the drawing, inwhich:

FIG. 1 is a side view of an embodiment having a test zone and a controlzone;

FIG. 2 is a side view of an embodiment having two test zones and acontrol zone;

FIG. 3 is a schematic view of the arrangement of test components on thetest strip prior to application of sample;

FIG. 4 is a schematic view of the test components after application ofsample, wherein movement of fluid and formation of complexes is shown inan example of a test that is negative for both penicillin specificallyand beta-lactams;

FIG. 5 is a schematic view of the test components after application ofsample, wherein movement of fluid and formation of complexes is shown inan example of a test that is positive for both penicillin specificallyand beta-lactams;

FIG. 6 is a schematic view of the test components after application ofsample, wherein movement of fluid and formation of complexes is shown inan example of a test that is negative for penicillin specifically andpositive for beta-lactams; and

FIG. 7 is a schematic view of the test components after application ofsample, wherein movement of fluid and formation of complexes is shown inan example of a test that is positive for penicillin specifically andnegative for beta-lactams.

DESCRIPTION OF EMBODIMENTS

In the following description, like reference characters designate likeor corresponding parts throughout the several views. Also in thefollowing description, it is to be understood that such terms as“forward,” “rearward,” “left,” “right,” “upwardly,” “downwardly,” andthe like are words of convenience and are not to be construed aslimiting terms. Referring now to the drawings in general, it will beunderstood that the illustrations are for the purpose of describingembodiments of the disclosure and are not intended to limit thedisclosure or any inventions thereto. Often, low pH substances, such asorange juice, have one or more analytes that may be detected asgenerally shown and described herein. To detect an analyte a binder forthe analyte can be employed, for instance the binder may be a bindingprotein, such as an enzyme, antibody, receptor or other substance,capable of binding to the analyte to form an analyte-binder complex. Thebinder, or analyte-binder complex, may be detected through variousmethods shown and described herein, including labeling the binder, and,therefore, the resulting complex, with a visible label, such as a goldparticle, and capturing the labeled complex with a capture agent. Hereinthe various embodiments provide unexpected advantages of detecting avariety of analytes, antibiotics, and the like, including, but notlimited to, oxytetracycline, or similar antibiotics, and streptomycin,or similar antibiotics, as understood by those skilled in the art havingthe benefit of this disclosure.

FIG. 1 illustrates one example of a test strip apparatus 8, comprised ofnitrocellulose membrane 3 and a binder application area 7 onto which thelabeled binders can be added. The binder application area can be, forexample, POREX® (POREX is a registered trademark of Porex TechnologiesCorp, Georgia USA), attached to solid support 2. If a sample pad 1 isused, the low pH sample is contacted to sample pad 1. Alternatively, thelow pH sample can be applied directly to the binder application area 7.If a sample pad 7 is used, sample flows from sample pad 1 to the sampleapplication area 7 containing, in an example, labeled specific binderand labeled multianalyte binder. Labeled specific binder and labeledmultianalyte binder will bind analyte from the sample and flow along thenitrocellulose membrane 3 to test zone 4. A portion of labeled specificbinder and labeled multianalyte binder, unbound by sample analyte, willbe captured at the related test zone. Remaining labeled binder, whetheror not bound by sample analyte, will flow to, and can bind to, controlzone 5. Some labeled binder may also flow past the control zone and intothe disposal pad 6. A stronger signal in the control zone as compared tothe test zone is a positive result. A weaker signal in the control zoneas compared to the test zone is a negative result. FIG. 2 shows anotherembodiment of test strip apparatus 8 further including a second testzone 9.

Prior to application to sample pad 1, the pH of a sample may need to beoptimized. For example, in cases where the sample has a pH less than 7,a buffer is used to increase the pH to the assay's optimal level.Samples, such as those taken from citrus juices, may have a pH anywherebetween about 2 and about 5. Those of ordinary skill in the art havingthe benefit of this disclosure will recognize additional sample andapplication uses. Typically, the buffer may comprise a mixture of acarrier protein and a base. The base may be a weak base such as sodiumbicarbonate. Bovine serum albumin may be a carrier protein used withinthe buffer. The low pH sample may have its pH optimized by combining thebuffer and low pH sample (ex: a 1:1 dilution ratio) prior to applyingthe sample onto sample pad 1. In one embodiment, the optimal level istypically a pH around 7, but may vary in other examples depending onassay components. Once the buffer is added, the sample may then beapplied to the test strip or the like.

FIG. 3 shows one example of test strip components of the embodimentshown in FIG. 2 prior to application of sample solution. The arrowsindicate the direction of sample flow from the binder application area7. Not shown is a sample pad 1 or other test solution application areathat may precede the binder application area 7. The binders in theapplication area are, in this example, labeled specific binder 21, inthis example labeled penicillin binder, and labeled multianalyte binder22, in this example labeled beta-lactam (BL) binder. The penicillinbinder 21 includes penicillin binding site 31, detectable label 32, forexample gold particle, and protein A binding site 33. The BL binder 22includes beta-lactam binding site 34, anti-BL binder binding site 35 andlabel 32. The test zone 9 includes immobilized penicillin 38. In thisembodiment, the beta-lactam ring of the immobilized penicillin isopened, depicted in this drawing as a break 24 in the ring, to reduce oreliminate the affinity of the beta-lactam binding site 34 of the BLbinder 22. Binding site 39 on immobilized penicillin 38 is available tocapture specific binder 21 unbound by penicillin from the sample. Thetest zone 4 includes immobilized representative beta-lactam 25 such asceforanide. BL binder binding site 37 is available to capture BL binder22 unbound by beta-lactam from the sample. The control zone 5 includesprotein A 41. The control zone 5 also includes anti-BL binder 42.Protein A can capture penicillin binder 21 through attachment to theprotein A site 33. As a result, protein A can capture both bound andunbound penicillin binder. If mixed together, Protein A 41 can also bindto anti-BL binder 42 at the control zone 5 to form an anti-BLbinder-protein A complex 43 that may retain the ability to capture bothpenicillin binder 21 and BL binder 22. Anti-BL binder 42 can capture BLbinder 22 unbound by beta-lactam from the sample or BL binder 22 boundby beta-lactam from the sample.

FIG. 4 shows a sample that is negative for both beta-lactams generallyand the specific beta-lactam penicillin at the appropriate level ofdetection. After application of the sample, test components typicallyflow out of binder application area 7. Penicillin binder 21, unbound bypenicillin from the sample, can be captured by penicillin immobilized onthe first test zone 9 to form a complex 51 that can be detected.Similarly, BL binder 22, unbound by beta-lactam from the test sample,can be captured by beta-lactam 25 immobilized on the second test zone 4to form complex 52 that can be detected. Protein A 41 in control zone 5,whether complexed to anti-BL binder or alone, can capture both bound andunbound penicillin binder 21 that flow past the test zone 9 withoutbeing captured. In this figure complex 45 includes penicillin binderunbound by penicillin from the sample and captured at the control zone.The anti-BL binder 42, whether complexed to protein A, or alone, cancapture both bound or unbound BL binder that flow past the test zone 4without being captured, for example to form complex 44 in the controlzone 5. In this figure some sample beta-lactam-BL binder complex 55 andsample penicillin-penicillin binder complex 54 are not captured andinstead flow to the disposal pad 6. Not all labeled binder, whether ornot bound by analyte from the sample, is necessarily captured at thetest zones or control zone. Remaining label can flow to the disposalzone 6. The negative result can be determined, in the drawing, bycounting the labels 32 in the control zone and test zones. In thisexample, the control zone 5 has two labels. The test zone 4 has fourlabels and the test zone 9 has four labels. Since the control zone hasfewer labels than either of the test zones the test is negative for bothpenicillin and other beta-lactams. Although one beta-lactam from thesample was captured by BL binder 22 to form a complex that was captured44 at the control zone 5, and one each of BL-binder and penicillinbinder were bound by antibiotic to form complex 55 and complex 54respectively, that flowed to the disposal zone 6, the semi-quantitativenature of the test is reflected in the negative result.

FIG. 5 shows a test that is positive for both penicillin specificallyand beta-lactams. Three penicillin binders 21 are bound at test zone 9,two BL binders 22 are bound at test zone 4 and five binders are bound atcontrol zone 5. Control zone 5 includes captured BL binder-label-samplebeta-lactam complex 44, captured sample penicillinbinder-label-penicillin complex 47 and captured unbound labeledpenicillin binder complex 45. Disposal zone 6 includes penicillin binder21 and BL binder-sample beta lactam complex 55, both of which slippedthrough uncaptured.

FIG. 6 shows a test that is positive for beta-lactams and negative forpenicillin specifically. Three labels are captured at the control zone 5and two labels are captured at test zone 4 while four labels arecaptured at test zone 9. Three labels are not captured and flow to thedisposal zone 6.

FIG. 7 shows a test that is negative for beta-lactams and positive forpenicillin specifically. Two labels are captured at the test zone 9 andfour labels are captured at the test zone 4 as compared to three labelscaptured at the control zone 5. Three labels are not captured and flowto the disposal zone 6.

It should be noted that the figures, particularly FIGS. 3-7, are highlysimplified depictions designed to exemplify both the various mechanismsof capture, binding and affinity and test result interpretation.Although in these figures the mechanisms are largely described relativeto detection of beta-lactams, including penicillin, the methods andassays described herein are not so limited.

In an example, a test zone capture agent can be, for example, ananalyte, representative analyte, or analyte analogue. The capture agentat some point must be immobilized to the strip so that it is eitherremoved from sample flow or is not solubilized by sample fluid flow.Immobilization on the strip, so that the capture agent is notsolubilized by fluid flow, can be accomplished using a carrier proteinsuch as bovine serum albumin (BSA), or other carrier protein well knownin the art, for example ovalbumin (OVA) or keyhole limpet hemocyanin(KLH).

Each test zone capture agent may capture all or a portion of the binder,from what is known as the mobile phase, which is not already bound withsample analyte. A binder that is bound by analyte from the sample tendsnot to be captured at the test zone. Binders that are not captured atthe test zone can be captured in the control zone or flow through to adisposal pad.

In an embodiment, a specific binder is a penicillin binder and amultianalyte binder is a BL binder. Both the penicillin binder and theBL binder can be detectably labeled, for example using gold particles.The labeled penicillin binder and labeled beta-lactam binder can becombined in a solution and applied, for example, by spraying, within orproximate to a pretreated POREX® (POREX is a registered trademark ofPorex Technologies Corp, Georgia USA) sample pad in contact with anitrocellulose membrane. The binders can also be combined with thesample in a container, such as a test tube, and added to the test stripwith the sample. When exposed to a sample such as orange juice, thepenicillin binder binds to penicillin in the orange juice and the BLbinder binds to beta-lactams (including to some extent penicillin) inthe orange juice to form complexes. Lateral capillary flow carries thecomplexes, and any uncomplexed labeled binders, to the test zone area ofthe strip.

In an embodiment using two binders, multiple test zones can be employedto capture the binders in separate zones. In an example in whichpenicillin binder and BL binder are used, the first test zone captureagent can include immobilized penicillin and the second test zonecapture agent can include an immobilized different beta-lactam, such asceforanide. In an embodiment in which the positions are reversed, thefirst test zone capture agent can include a representative beta-lactamand the second test zone capture agent can include penicillin. In thetest zones, the capture agents can capture the binders that have notbeen previously bound by sample analyte. Such attachment at the testzone can generate a visible signal when a detectable label, such as goldor other label well known in the art, is used. Other particles that maybe useful include, but are not limited to, colloidal sulphur particles;colloidal selenium particles; colloidal barium sulfate particles;colloidal iron sulfate particles; metal iodate particles; silver halideparticles; silica particles; colloidal metal (hydrous) oxide particles;colloidal metal sulfide particles; colloidal lead selenide particles;colloidal cadmium selenide particles; colloidal metal phosphateparticles, colloidal metal ferrite particles, any of the above-mentionedcolloidal particles coated with an organic or inorganic layer; proteinor peptide molecules; liposomes; or organic polymer latex particles,such as polystyrene latex beads. Other labels may also be usefulincluding, but not limited to, luminescent labels; fluorescent labels;or chemical labels, such as electroactive agents (e.g., ferrocyanide);enzymes; radioactive labels; or radiofrequency labels.

In an embodiment, two control zone capture agents are employed withinthe same control zone. The capture agents can each capture a differentbinder. In an embodiment in which a penicillin binder and a BL binderare both employed, protein A can be one control zone capture agent andan antibody to beta-lactam binder (“anti-BL binder”) can be the othercapture agent. In embodiments in which the anti-BL binder also hasprotein A binding affinity, such as when the anti-BL binder is rabbitantibody, the two control zone capture agents may become linked. Inaddition, either or both the protein A and anti-BL binder can beimmobilized on the control zone using a carrier protein such as BSA.

In another embodiment, one control zone capture agent can be attached atthe control zone and another control zone capture agent can be appliedlater for example by flowing to the control zone with the sample. Forexample, when the binders are a penicillin binder and a BL binder, thecontrol zone can include protein A and anti-BL binder. Alternatively,the control zone can include protein A and the anti-BL binder can bebound to the BL binder and added to the strip with the BL binder such ason the sample pad. BL binder will retain beta-lactam binding activityafter binding to anti-BL binder, but not after binding to a beta-lactamdrug in the sample. In such an embodiment, the control zone captureagent is selected for its affinity to the anti-BL binder at a site notoccupied by BL binder. Examples of such capture agents include ProteinA, protein G, recombinant protein AG or other substances such assubstances that bind, for example, to the constant region of anantibody.

In an embodiment, detectably labeled multianalyte binder (“labeledmultianalyte binder) and detectably labeled specific binder (“labeledspecific binder”) are combined with unlabeled binders, such as unlabeledantibodies. The unlabeled antibodies are selected for their affinity toantibiotics to which the other binders, for example the multianalytebinder, are oversensitive. Such unlabeled binders compete with labeledmultianalyte binders for a specific antibiotic and, thereby, reduce testsensitivity to the antibiotic/analyte to which the unlabeled bindershave affinity. The unlabeled binders can have affinity for some or allof the analyte to which the labeled multianalyte binder has affinity. Byincluding unlabeled binders with affinity for some, but not all, of theanalytes to which a competing labeled binder has affinity, sensitivityof the test to those selected antibiotics/analytes will be reduced.

In some embodiments there is excess capture reagent in the control zone.Excess capture reagent will provide consistent control zone detection.In other control zone embodiments there is an excess of the generalantibody binder, such as protein A, relative to the multianalyte bindercapture agent, such as anti-BL binder, such as rabbit antireceptor. In aparticular example using protein A and rabbit anti-BL binder, thecontrol zone will have sufficient excess protein A available forcapturing penicillin binder and binding the anti-BL binder.

In certain embodiments, in a negative sample the control zone willcapture fewer labels as compared to any one of the test zones, in apositive sample for a single analyte or family, the control zone willcapture more labels than one of the test zones and fewer labels than theother and, if positive for all analytes, each of the test zones willcapture fewer labels as compared to the control zone.

Including multiple capture agents at the control zone, such as bothprotein A, or other general antibody binder, and specific antibody to abinder, such as anti-BL binder, provides the possibility of a test stripfor detection of multiple analytes such as multiple antibiotics. Forexample, a test strip to detect sulfonamides, tetracyclines, amphenicolsor macrolides, possibly combined on a test strip to detect beta-lactams,with a single control zone for comparison to multiple test zones for thedifferent antibiotics. A control zone can have multiple specificantibodies to particular binders, multiple multianalyte binderantibodies, multiple antibody binding proteins or combinations thereof.Similarly, the control zone can include multiple antibodies of differenthost species and not necessarily include a general antibody binder. Forexample, if one binder is a mouse monoclonal antibody and another is arabbit polyclonal antibody, the control zone can include an anti-mouseantibody and an anti-rabbit antibody.

In another embodiment, the assay can be in the form of a so-calledsandwich assay using different types of binders, such as a combinationof polyclonal and monoclonal antibodies or a combination of differentspecies polyclonal antibodies. Tests to detect multiple analytes using asandwich assay can include a plurality of labeled analyte binders thatbind with different analytes. The test zone can include multipleimmobilized binders for the analyte, for example with affinity todifferent regions of the analyte to which the labeled antibody hasaffinity. The control zone could include a combination of immobilizedcapture agents including, for example, combinations of anti-speciesantibodies for reactions to each of the different analyte binders. Thecontrol zone could also combine, depending on the binders used, acombination of general antibody binding protein, such as protein A, andan antibody for one of the binders to which protein A does not haveadequate affinity, such as anti-mouse antibody. In such an embodiment,as with the other embodiments described herein in which the non-sandwichformat is used, either the analyte binder can be labeled with a visiblelabel or, in an embodiment, the label can require a further reactionsuch as when the label is an enzyme or substrate of an enzyme linkedimmunosorbent assay (ELISA).

In another embodiment, one type of binder, for example a labeledpolyclonal antibody, can be used to detect a single analyte or a groupof related analytes. One test zone and one control zone can be used tocapture the labeled binder. The control zone can include multiplecapture agents, each with different affinity to the binder, such as eachwith affinity to different parts of the binder. For example, if thelabeled antibody is a rabbit polyclonal, the control zone capture agentscan include, for example, an anti-rabbit antibody and an antibodybinding protein such as protein A. By including different control zonecapture agents, it may be possible to improve binder capture at thecontrol zone and, thereby, improve the control zone signal.

In some embodiments BL binder is isolated directly from, for example,B.st., by immobilized ligand affinity chromatography techniques that arewell known in the art.

The binder can also be expressed from other hosts by inserting into thehost genome the sequence of the BL binder from B.st.

Another example of a useful multianalyte binder, that can be used incertain embodiments, includes a beta-lactam binding protein isolatedfrom Bacillus lichenformis. Other possibly useful binders includemacromolecules, such as monoclonal or polyclonal antibodies, hormonereceptors and enzymes and synthetic receptors such as those generatedthrough molecular imprinting of synthetic polymers or molecularimprinting inside dendrimers.

Numerous characteristics and advantages have been set forth in theforegoing description, together with details of structure and function.Many of the novel features are pointed out in the appended claims. Thedisclosure, however, is illustrative only, and changes may be made indetail, especially in matters of shape, size, and arrangement of parts,within the principle of the disclosure, to the full extent indicated bythe broad general meaning of the terms in which the general claims areexpressed. It is further noted that, as used in this application, thesingular forms “a,” “an,” and “the” include plural referents unlessexpressly and unequivocally limited to one referent.

We claim:
 1. A method for detecting one or more analytes in a solutioncomprising: applying a binder to a test strip, the binder configured forgenerating a detectable signal and capable of combining with the analyteto form a binder-analyte complex; immobilizing, within one or more testzones of the test strip, at least one test zone capture agents capableof capturing the binder; immobilizing, within a control zone of the teststrip, at least one control zone capture agent adapted for capturing thebinder and the binder-analyte complex; providing a sample having a pHless than 7; raising the pH of the sample by diluting with a buffercomprising a carrier protein and a base to form the solution; adding thesolution to the test strip; and providing a test result by comparing asignal in the control zone to a signal in the test zone, and wherein oneor more analytes in the solution, when present, are adapted to combinewith the at least one binder to form the binder-analyte complex.
 2. Themethod of claim 1, wherein the sample is a citrus juice.
 3. The methodof claim 1, wherein the pH of the sample is between about 2 and about 5.4. The method of claim 1, wherein the method includes detecting anoxytetracycline antibiotic.
 5. The method of claim 1, wherein the methodincludes detecting a streptomycin antibiotic.